Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS.

Julia Nagy,Finn Werner,Sarah Schulz,Katherine Smollett,Alan C.M Cheung,Dina Grohmann,Jens Michaelis

doi:10.1038/ncomms7161

Abstract

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

Highlights

Transcription of all cellular genomes is carried out by evolutionary related multisubunit RNA polymerases (RNAPs)
Labelled DNA oligonucleotides, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and RNAP were combined to yield a large network of B70 differently labelled complexes, each with a single single-molecule Forster resonance energy transfer (smFRET) pair at a desired location (Fig. 1a,b)
The smFRET and global Nano-Positioning System (NPS) data presented here reveal the complete architecture of the open promoter complex in archaea including the paths of the non-template DNA (ntDNA) and template DNA strand (tDNA) strands, and the location of the three transcription initiation factors TBP, TFB and TFE

Summary

Introduction

Transcription of all cellular genomes is carried out by evolutionary related multisubunit RNA polymerases (RNAPs). TBP and TFIIB assemble at the promoter[4] and recruit Pol II as well as other factors to form the preinitiation complex (PIC) This complex is referred to as the closed complex (CC), which subsequently undergoes large conformational rearrangements during which the DNA strands are separated and the template DNA strand (tDNA) is loaded into the RNAP active site to form the open complex (OC). The archaeal transcription apparatus is an excellent model system for the eukaryotic Pol II system[15] as its RNAP and associated basal transcription factors are homologous, and because the entire system from hyperthermophilic archaea can be reconstituted from recombinant proteins[16] This enables us to site introduce mutations or molecular probes such as fluorescent dyes for single-molecule fluorescence analysis[17,18]. Single-molecule techniques have shown great potential to resolve the dynamics of transcription processes because they allow for the direct and real-time observation of transcription, one molecule at a time[23]

Methods

Results

Conclusion